WO2025222151A1 - Components and methods for making scaffold tissues via pectin-containing sacrificial templates - Google Patents

Abstract

Disclosed herein are aspects of a scaffold for tissue regeneration, the scaffold comprising a first component comprising one or more biocompatible materials; and a second component comprising a pectin-containing sacrificial template, wherein the second component is intermittently associated with the first component. In certain aspects, the scaffold may further comprise a disassociating agent for dissolving the second component. Also, disclosed herein is a method for making a scaffold for tissue regeneration, the method comprising providing a first feed to a first nozzle and a second feed to a second nozzle, the first feed comprising one or more biocompatible materials, the second feed comprising pectin, pectin derivatives, or a combination thereof; and obtaining a composition comprising a first component and a second component, the first component comprising the one or more biocompatible materials, and the second component comprising a pectin-containing sacrificial template that is intermittently associated with the first component.

Description

COMPONENTS AND METHODS FOR MAKING SCAFFOLD TISSUES VIA A PECTIN-CONTAINING SACRIFICIAL TEMPLATE

CROSS-REFERENCE TO RELATED APPLICATION

[001] This application claims the benefit of U.S. Provisional Application No. 63/636,442, filed on April 19, 2024, which is incorporated herein in its entirety.

FIELD

[002] The present disclosure concerns a scaffold and method for making a scaffold for tissue regeneration applications.

BACKGROUND

[003] Traditional approaches in making scaffold tissues for tissue regeneration applications typically exhibit low mechanical strength and low porosity. These scaffolds have a low biocompatibility with host tissue and a high morbidity rate (e.g., 3D-printed scaffolds used in treating bone defects in musculoskeletal and craniofacial tissue caused by disease and tumor resection). Moreover, the porous structure of the 3D-printed scaffolds comes at the cost of reducing the mechanical strength of the scaffold tissues. Therefore, 3D-printed scaffolds produced using traditional approaches do not result in the structural, functional, and biological features of native biological tissue. Accordingly, there is a need in the art for novel approaches that can produce scaffold tissue with hierarchical heterogenous structure and predefined desirable mechanical properties that can be integrated with native biological tissue.

SUMMARY

[004] Disclosed herein are various compositions and methods for forming structures that can be used in tissue regeneration applications.

[005] For example, in one aspect, a scaffold for tissue regeneration applications can comprise a first component that includes one or more biocompatible materials, and a second component that includes a pectin-containing sacrificial template. The second component can be intermittently associated with the first component. [006] In another aspect, a method for making a scaffold for tissue regeneration can include providing a first feed to a first nozzle and a second feed to a second nozzle and obtaining a composition comprising a first component and a second component. The first feed can include one or more biocompatible materials, and the second feed can include pectin, pectin derivatives, or a combination thereof. The first component can include one or more biocompatible materials, and the second component can include a pectin-containing sacrificial template that is intermittently associated with the first component. In some examples, the method can also include introducing a disassociating agent to disassociate the pectin-containing sacrificial template from the first component.

[007] The foregoing and other objects, features, and advantages of the present disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[008] FIG. 1 is a schematic diagram illustrating at least certain aspects of a method according to aspects of the present disclosure.

[009] FIG. 2A is a modulus (Pa) versus shear strain (%) graph obtained from the rheological analysis of pectin slurry (pectin ink) with a concentration of 10%, 17.5%, and 25% dissolved in water; demonstrating that the storage modulus (G) and loss modulus (G’) remained constant for the given amplitude at the different concentrations.

[010] FIG. 2B is a modulus (Pa) versus frequency (Hz) graph obtained from the rheological analysis of pectin slurry (pectin ink) with a concentration of 10%, 17.5%, and 25% dissolved in water; demonstrating that the storage modulus (G) and loss modulus (G’) remained constant for the given frequency at the different concentrations.

[011] FIG. 2C is a viscosity (mPa.s) versus shear rate (1/s) graph obtained from the rheological analysis of pectin slurry (pectin ink) with a concentration of 10%, 17.5%, and 25% dissolved in water; demonstrating shear thinning behavior (i.e., as the shear rate increased, the viscosity decreased) at the different concentrations.

[012] FIG. 2D is a modulus (Pa) versus temperature (°C) gradient graph obtained from the rheological analysis of pectin slurry (pectin ink) with a concentration of 10%, 17.5%, and 25% dissolved in water; demonstrating pectin having fluid- like behavior up to 70 °C at the different concentrations.

[013] FIG. 3A is an image of a 6-well plate each comprising a 3D-printed scaffold (10% pectin) subjected to ethanol curing concentrations of 100% (top-left plate), 75% (top-middle plate), 50% (top-right plate), 25% (bottom-left plate), and 0% (bottom-middle plate); demonstrating a desirable consistency and stiffness as the ethanol curing concentration increased.

[014] FIG. 3B is an image of a 6-well plate each comprising a 3D-printed scaffold (15% pectin) subjected to an ethanol curing concentrations of 100% (top-left plate), 75% (top-middle plate), 50% (top-right plate), 25% (bottom-left plate), and 0% (bottom-middle plate); demonstrating a desirable consistency and stiffness as the pectin concentration and ethanol curing concentration increased.

[015] FIG. 3C is an image of a 6-well plate each comprising a 3D-printed scaffold (20% pectin) subjected to an ethanol curing concentrations of 100% (top-left plate), 75% (top-middle plate), 50% (top-right plate), 25% (bottom-left plate), and 0% (bottom-middle plate); demonstrating a desirable consistency and stiffness as the pectin concentration and ethanol curing concentration increased.

[016] FIG. 3D is an image of a 6-well plate each comprising a 3D-printed scaffold (25% pectin) subjected to an ethanol curing concentrations of 100% (top-left plate), 75% (top-middle plate), 50% (top-right plate), 25% (bottom-left plate), and 0% (bottom-middle plate); demonstrating a desirable consistency and stiffness as the pectin concentration and ethanol curing concentration increased.

[017] FIG. 4 is a line graph of the degradation of 3D-printed scaffold comprising a pectin- containing sacrificial template (%) versus time (minutes) showing the degradation of 3D-printed scaffolds comprising a pectin-containing sacrificial template and supporting that the formation of porous channels in the hydroxyapatite (HA)/polyvinyl butyral (PVB) scaffold arises by exhibiting that the pectin associates and degrades into the scaffold.

[018] FIGS. 5 A-5I are images of a well plate comprising a 3D-printed scaffold comprising a pectin-containing sacrificial template showing the progressive degradation of the pectin structure at 0 minutes (FIG. 5A), 5 minutes (FIG. 5B), 10 minutes (FIG. 5C), 15 minutes (FIG. 5D), 20 minutes (FIG. 5E), 25 minutes (FIG. 5F), 30 minutes (FIG. 5G), 35 minutes (FIG. 5H), and 40 minutes (FIG. 51); demonstrating the formation of porous channels in the HA/PVB scaffold as pectin associates and degrades into the scaffold. [019] FIG. 6A is an image of a 3D-printed scaffold comprising a pectin-containing sacrificial template, which demonstrates the integration of the pectin-containing sacrificial template.

[020] FIG. 6B is an image of two 3D-printed triangular shaped scaffolds and two 3D-printed circular shaped scaffolds comprising a pectin-containing sacrificial template, which demonstrates the integration of the pectin-containing sacrificial template and the printing of different geometric shapes.

[021] FIG. 6C is a microscopy image of a 3D-printed scaffold comprising a pectin-containing sacrificial template and demonstrating the integration of the pectin-containing sacrificial template.

[022] FIGS. 6D-6E are microscopy images of a 3D-printed scaffold comprising a pectin- containing sacrificial template after the immersion in water (FIG. 6D) and after 6 hours (FIG. 6E) demonstrating the network of porous channels inside the HA/PVB scaffold generated by the dissolved pectin-containing sacrificial template.

[023] FIG. 6F is a microscopy image showing the cross-section of the 3D-printed scaffold comprising a pectin-containing sacrificial template post-disintegration in water and demonstrating desirable integration and subsequent dissolution of the pectin-containing sacrificial template.

[024] FIG. 7 A is a microscopy image showing a 3D-printed scaffold comprising a pectin- containing sacrificial template arranged in a linear pattern.

[025] FIG. 7B is a microscopy image of the 3D-printed scaffold of FIG. 7A showing the dissociation of the pectin-containing sacrificial template.

[026] FIG. 7C is a microscopy image showing a 3D-printed scaffold comprising a pectin- containing sacrificial template arranged in a sinuous pattern.

[027] FIG. 7D is a microscopy image of the 3D-printed scaffold of FIG. 7C showing the dissociation of the pectin-containing sacrificial template.

[028] FIG. 7E is a microscopy image showing a 3D-printed scaffold comprising a pectin- containing sacrificial template arranged in a chevron pattern.

[029] FIG. 7F is a microscopy image of the 3D-printed scaffold of FIG. 7E showing the dissociation of the pectin-containing sacrificial template. [030] FIG. 8A is a scanning electron microscope (SEM) image showing a top view of a scaffold comprising HA constructed without a pectin-containing sacrificial template.

[031] FIG. 8B is a SEM image showing a cross-sectional view of a scaffold comprising HA constructed without a pectin-containing sacrificial template.

[032] FIG. 8C is a SEM image showing a top view of a scaffold comprising HA and a pectin- containing sacrificial template (pre-degradation).

[033] FIG. 8D is a SEM image showing a cross-sectional of a scaffold comprising HA constructed with a pectin-containing sacrificial template (pre-degradation).

[034] FIG. 8E is a SEM image showing a top view of a degraded scaffold comprising HA constructed with a pectin-containing sacrificial template.

[035] FIG. 8F is a SEM image showing a cross-sectional view of a degraded scaffold comprising HA constructed with a pectin-containing sacrificial template.

[036] FIG. 9 is a graph showing the Young's modulus of a scaffold comprising an HA component and a pectin-containing sacrificial template (HA-Pectin), a degraded scaffold comprising an HA component constructed with a pectin-containing sacrificial template (HA-Degraded Pectin), a scaffold comprising native HA (HA), and a scaffold comprising a native pectin-containing sacrificial template HA-Pectin (Pectin).

[037] FIG. 10A is a bar graph showing the percentage of endothelial cell attachment to a scaffold comprising an HA component (HA); a scaffold comprising a P-TCP component (P-TCP); a degraded scaffold comprising an HA component constructed with a pectin-containing sacrificial template (HA-degraded Pectin); and a degraded scaffold comprising a P-TCP component constructed with a pectin-containing sacrificial template (P-TCP-degraded Pectin) after four hours.

[038] FIG. 10B is a bar graph showing the percentage of osteoblast cell attachment to a scaffold comprising an HA component (HA), a scaffold comprising a P-TCP component (P-TCP), a degraded scaffold comprising an HA component constructed with a pectin-containing sacrificial template (HA-degraded Pectin), and a degraded scaffold comprising a P-TCP component constructed with a pectin-containing sacrificial template (P-TCP-degraded Pectin) after four hours. [039] FIG. 11 A is a microscopy image showing the percentage of endothelial cell viability on a scaffold comprising an HA component using live/dead staining.

[040] FIG. 1 IB is a microscopy image showing the percentage of endothelial cell viability on a degraded scaffold comprising an HA component constructed with a pectin-containing sacrificial template component using live/dead staining.

[041] FIG. 11C is a microscopy image showing the percentage of endothelial cell viability on a scaffold comprising an [>-TCP component using live/dead staining.

[042] FIG. 1 ID is a microscopy image showing the percentage of endothelial cell viability on a degraded scaffold comprising a p-TCP component constructed with a pectin-containing sacrificial template component using live/dead.

[043] FIG. 1 IE is a bar graph showing the quantitative data of the endothelial cell viability on the scaffold comprising an HA component (HA) of FIG. 11 A, the degraded scaffold comprising an HA component constructed with a pectin-containing sacrificial template (HA-degraded Pectin) of FIG.

1 IB; the scaffold comprising a P-TCP component (P-TCP) of FIG. 11C; and the degraded scaffold comprising a P-TCP component constructed with a pectin-containing sacrificial template (0-TCP- degraded Pectin) of FIG. 1 ID.

[044] FIG. 12A is a microscopy image showing the percentage of osteoblast cell viability on a scaffold comprising an HA component using live/dead staining.

[045] FIG. 12B is a microscopy image showing the percentage of the osteoblast cell viability on a degraded scaffold comprising an HA component constructed with a pectin-containing sacrificial template component using live/dead staining.

[046] FIG. 12C is a microscopy image showing the percentage of osteoblast cell viability on a scaffold comprising an p-TCP component using live/dead staining.

[047] FIG. 12D is a microscopy image showing the percentage of osteoblast cell viability on a degraded scaffold comprising a P-TCP component constructed with a pectin-containing sacrificial template component using live/dead staining.

[048] FIG. 12E is a bar graph showing the quantitative data of the osteoblast cell viability on the scaffold comprising an HA component (HA) of FIG. 12A, the degraded scaffold comprising an HA component constructed with a pectin-containing sacrificial template (HA-degraded Pectin) of FIG. 12B; the scaffold comprising a 0-TCP component (0-TCP) of FIG. 12C; and the degraded scaffold comprising a [3-TCP component constructed with a pectin-containing sacrificial template (0-TCP- degraded Pectin) of FIG. 12D.

[049] FIG. 13A is a confocal microscopy image (lOx magnification) showing endothelial cells with fluorescence staining (DAPI for nuclei and Phalloidin for actin filaments) present on a degraded scaffold comprising an HA component constructed with a pectin-containing scaffold template arranged in a linear pattern.

[050] FIG. 13B is a confocal microscopy image (lOx Sora 2.8x magnification) showing endothelial cells with fluorescence staining (DAPI for nuclei and Phalloidin for actin filaments) present on a degraded scaffold comprising an HA component constructed with a pectin-containing scaffold template arranged in a linear pattern.

[051] FIG. 13C is a confocal microscopy image (lOx magnification) showing osteoblasts with fluorescence staining (DAPI for nuclei and Phalloidin for actin filaments) present on a degraded scaffold comprising an HA component constructed with a pectin-containing scaffold template arranged in a linear pattern.

[052] FIG. 13D is a confocal microscopy image (lOx magnification Sora 2.8x) showing osteoblasts with fluorescence staining (DAPI for nuclei and Phalloidin for actin filaments) present on a degraded scaffold comprising an HA component constructed with a pectin-containing scaffold template arranged in a linear pattern.

DETAILED DESCRIPTION

I. Overview of Terms, Ranges, and Definitions

[053] The following explanations of terms are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. As used herein, “comprising” means “including” and the singular forms “a” or “an” or “the” include plural references unless the context clearly dictates otherwise. The term “or” refers to a single element of stated alternative elements or a combination of two or more elements unless the context clearly indicates otherwise.

[054] The methods described herein should not be construed as limiting in any way. Instead, the present disclosure is directed toward all novel and non-obvious features and aspects of the present disclosure, alone and in various combinations and sub-combinations with one another. The disclosed methods are not limited to any specific aspect or feature or combinations thereof, nor do the disclosed methods require that any one or more specific advantages be present or problems be solved. Any theories of operation are to facilitate explanation, but the methods are not limited to such theories of operation.

[055] Although the operations of some of the disclosed methods are described in a particular, sequential order for convenient presentation, it should be understood that this manner of description encompasses rearrangement, unless a particular ordering is required by specific language set forth below. For example, operations described sequentially may in some cases be rearranged or performed concurrently. Moreover, for the sake of simplicity, the attached figures may not show the various ways in which the disclosed devices and methods can be used in conjunction with other devices and methods. Additionally, the description sometimes uses terms like “produce” and “provide” to describe the disclosed methods. These terms are high-level abstractions of the actual operations that are performed. The actual operations that correspond to these terms will vary depending on the particular implementation and are readily discernible by one of ordinary skill in the art. Furthermore, examples may be described with reference to directions indicated as “above,” “below,” “upper,” “lower,” and the like. These terms are used for convenient description, but do not imply any particular spatial orientation unless so indicated.

[056] In some examples, values, procedures, or devices may be referred to as “lowest,” “best,” “minimum,” or the like. It will be appreciated that such descriptions are intended to indicate that a selection among many used functional alternatives can be made, and such selections need not be better, smaller, or otherwise preferable to other selections.

[057] Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting, unless otherwise indicated. Other features of the disclosure are apparent from the following detailed description and the claims.

[058] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, percentages, temperatures, times, and so forth, as used in the specification or claims are to be understood as being modified by the term “about.” Accordingly, unless otherwise indicated, implicitly or explicitly, the numerical parameters set forth are approximations that can depend on the desired properties sought and/or limits of detection under standard test conditions/methods.

When directly and explicitly distinguishing embodiments from discussed prior art, the embodiment numbers are not approximates unless the word “about” is recited. Furthermore, not all alternatives recited herein are equivalents.

[059] The following terms and definitions are provided:

[060] Disassociating Agent: Refers to a liquid solvent, gaseous solvent, an environment comprising one or more temperature differential, or any combination thereof, used for disassociating and removing a sacrificial template from one or more components comprising one or more biocompatible materials. Removing can be, but is not limited to, dissolving, and melting.

[061] Intermittently Associated: Refers to the temporary coupling of a component comprising a sacrificial template that is deposited on one or more components comprising one or more biocompatible materials, and later removed from the one or more components comprising one or more biocompatible materials leaving behind a three-dimensional structure having interconnected channels and/or pores.

[062] Sacrificial Template: Refers to a removable structure that is deposited on one or more components comprising one or more biocompatible materials. The sacrificial template can be configured to form interconnected channels and/or pores in a scaffold. IL Introduction

[063] Conventional compositions and methods used in 3D-printing scaffold fail to achieve a proper balance between porosity and mechanical strength, which results in poor biocompatibility. Thus, there is a need in the art for 3D-printed scaffolds that are compatible with biological native tissue while also providing structural support.

[064] Components and methods disclosed herein include a customizable composition that achieves biocompatibility with native biological tissue and has desirable porosity and mechanical strength. Typically, the customizable composition comprises a sacrificial template comprising pectin, which allows for the controlled formation of channels within the scaffold, and thereby forms well-interconnected pore structures. Control over pore size and interconnectivity promotes cell growth and tissue formation. Moreover, the sacrificial template is printed on a primary component comprising biocompatible materials for tissue engineering applications.

[065] In certain aspects, the present disclosure can produce 3D-printed scaffolds for use in bone tissue engineering. Control over the pore size and interconnectivity of the tissue architecture creates tailored microenvironments that increase cell distribution and functionality in tissues such as in, but not limited to, cartilage and soft tissue engineering. Furthermore, the incorporation of endothelial cells with sacrificial templates disclosed herein can support angiogenesis, which can be used for engineering vascularized structures that mimic natural tissue conditions. Moreover, the channels can be loaded with therapeutic agents, growth factor, pharmaceutical compositions, and provide for the controlled release of the therapeutic agents, growth factor, pharmaceutical compositions as the scaffold degrades.

III. Composition

[066] Aspects of the present disclosure are directed to a composition for tissue regeneration. The composition can comprise a first component comprising one or more biocompatible materials; and a second component comprising a pectin-containing sacrificial template, wherein the second component is intermittently associated with the first component. The first component can provide structural support to define the overall shape of the scaffold. The pectin-containing sacrificial template can be disassociated to form pores and/or channels within the degraded scaffold. The pores in the degraded scaffold provide for cell adhesion, distribution, proliferation and differentiation, and matrix secretion. For example, the pores in the degraded scaffold can provide for the flow of nutrients, cells, or other substances that affect cell distribution, tissue infiltration, bone tissue regeneration, angiogenesis, or any combination thereof.

[067] In certain aspects, the one or more biocompatible materials comprise one or more synthetic biodegradable polymers, one or more calcium phosphate-containing compounds, one or more cellulose-based polymers, or any combination thereof. In some aspects, the synthetic polymer can comprise be for example, but is not limited to, polyvinyl butyral (PVB), polycaprolactone (PCL), polyvinyl alcohol (PVA), polylactic-co-glycolic acid (PLGA), or any combination thereof. In aspects disclosed herein, the calcium phosphate can be a bioceramic such as, but not limited to, hydroxyapatite, oxy apatite, tetracalcium phosphate (TTCP), dicalcium phosphate anhydrous (DCPA), P-tricalcium phosphate (P-TCP), a-tricalcium phosphate (a-TCP), calcium phosphate cement (CPC). In certain aspects, the first component may further comprise water insoluble polymers such as, but not limited to, collagen, chitosan, alginate, or any combination thereof. In particular aspects disclosed herein, the first component may further comprise one or more metals, such as, but is not limited to, titanium.

[068] In aspects disclosed herein, the second component may comprise solid particulates, polymeric structures, or a combination thereof. The solid particulates, polymeric structures, or combinations thereof, can disassociate from the first component when subjected to dissociating agents comprising liquid solvents, gaseous solvent, and/or based on a temperature differentials in the environment. In some aspects, the composition for tissue regeneration may further comprise a disassociating agent for disassociating the second component from the first component, wherein the first component is insoluble in the disassociating agent. In one preferable aspect, the disassociating agent can be an aqueous solvent for dissolving the second component comprising a pectin- containing sacrificial template.

[069] In some aspects, the solid particulates, polymeric structures, or a combination thereof, form a sacrificial template comprising pectin, a pectin derivative, or a combination thereof. The pectin and pectin derivatives can be for example, but are not limited to, pectin methacrylate, low methoxyl pectin (LMP), high methoxyl pectin, (HMP), pectin maleate, pectin tartrate, pectin phthalate, fruit pectin, or any combination thereof.

[070] In particular aspects disclosed herein, the second component may further comprise a surfactant. Pectin can be used in combination with the surfactant to achieve a roundness in the printed pectin filaments, which can form the rounded channel networks that accelerate the supply of oxygen and/or nutrients to promote cell growth. In some aspects, the surfactant can be for example, but is not limited to, pluronic, polyvinyl alcohol (PVA), polyvinyl butyral (PVB), polycaprolactone (PCL), polyethylene glycol (PEG), or any combination thereof. In certain aspects the pluronic can be, but is not limited to, Pluronic F-127, Pluronic F-108, Pluronic F-68, Pluronic P-123.

[071] In some aspects, the second component comprising a pectin-containing sacrificial template can be used to encapsulate one or more active agents. In certain aspects, the active agent can be, but is not limited to, a pharmaceutical composition, a growth factor, a protein, an antibody, antibody fragments, peptides, oligonucleotides, plasmid DNA, plasmid RNA, or any combination thereof. In some aspects, the second component comprising a pectin-containing sacrificial template may further comprise one or more water insoluble polymers such as, but not limited to, chitosan, gelatin, alginate, or any combination thereof. Without being bound by one theory of operation, the one or more water insoluble polymers can delay the degradation of the pectin-containing sacrificial template and thus control the degradation process and facilitate the controlled release of the one or more active agents.

[072] In aspects disclosed herein, the composition can be used as an ink for 3D-printing a scaffold for tissue regeneration applications. In certain aspects, the ink may further comprise one or more cells for tissue regeneration. The one or more cells can be for example, but are not limited to, osteoblasts (bone cells), chondrocytes (cartilage cells), adipocytes (fat cells), fibroblasts, keratinocytes, endothelial cells, smooth muscle cells, hepatocytes, cardiomyocytes, neural stem cells (NSCs), neural progenitor cells (NPCs), or any combination thereof.

[073] In certain aspects, the ink can form a 3D-printed scaffold that controllably releases one or more active agents administered to a subject to effect a change, such as treatment, amelioration, or prevention of a disease or disorder or at least one symptom associated therewith. In particular aspects disclosed herein, pectin can encapsulate one or more active agents. In certain aspects, pectin can encapsulate bone morphogenetic proteins (BMPs), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-0). In particular aspects disclosed herein, pectin can encapsulate pharmaceutical compositions such as, but not limited to, antibiotic drugs (e.g., Gentamicin, Vancomycin, Tobramycin, Ciprofloxacin, Doxycycline, Erythromycin, Clindamycin), analgesic drugs and anti-inflammatory drugs (e.g., Ibuprofen, Lidocaine, Paracetamol, Morphine, Oxycodone, Fentanyl). IV. Method of Making

[074] Aspects of the present disclosure also concern a method for making a scaffold for tissue regeneration applications. The method may comprise obtaining the first component comprising one or more biocompatible materials and producing a second component comprising the pectin- containing sacrificial template above the first component, wherein the pectin-containing sacrificial template is intermittently associated with the first component.

[075] In aspects disclosed herein, a disassociating agent can be introduced to disassociate the pectin-containing sacrificial template, wherein the first component is insoluble in the disassociating agent. In certain aspects, a liquid solvent or gaseous solvent can be introduced to dissolve the pectin-containing sacrificial template. In particular aspects disclosed herein, a temperature differential can be introduced to disassociate the pectin-containing template. In a preferred aspect, the disassociating agent is an aqueous solvent. In certain aspects, the first component comprising the one or more biocompatible materials can be dispersed in alcohol and a second component comprising the pectin-containing sacrificial template is cured by the alcohol of the first component.

[076] In some aspects, the method may comprise providing a first feed to a first nozzle, wherein the first nozzle extrudes a slurry comprising the first component comprising one or more biocompatible materials. In aspects disclosed herein, the first nozzle can have a nozzle size ranging from greater than 0 mm to 5 mm, such as from 0.030 mm to 4 mm, 0.030 mm to 3 mm, 0.030 mm to 2 mm, 0.030 mm to 1.8 mm, 0.030 mm to 1.6 mm, 0.030 mm to 1.4 mm, 0.030 mm to 1.2 mm, 0.030 mm to 1 mm. In certain aspects, the first nozzle can have a printing speed ranging from greater than 0 mm/s to 200 mm/s, such as from 5 mm/s to 200 mm/s, 5 mm/s to 175 mm/s, 5 mm/s to 150 mm/s, 5 mm/s to 125 mm/s, 5 mm/s to 100 mm/s, 5 mm/s to 75 mm/s, 5 mm/s to 50 mm/s, 5 mm/s to 25 mm/s. In particular aspects disclosed herein, the first nozzle can have a pressure of 1 bar, 2 bar, 3 bar, 4 bar, or 5 bar. In some aspects, the temperature of the first feed have a temperature ranging from -20 °C to 100°C, such as from -10 °C to 90°C, -10 °C to 85 °C, -10 °C to 80 °C, -10 °C to 75 °C, -10 °C to 70 °C, -10 °C to 60 °C, -10 °C to 50 °C, -10 °C to 40 °C, -10 °C to 35 °C, -10 °C to 30 °C, -10 °C to 25 °C, -10 °C to 20 °C, or -10 °C to 15°C.

[077] Typically, the first component comprises calcium phosphate and a synthetic polymer. In certain aspects, the first feed may comprise a synthetic polymer having a concentration ranging from 1% weight by total volume of the total composition (w/v) of the solution to 70% w/v, such as from 10% w/v to 70% w/v, 20% w/v to 70% w/v, 30% w/v to 70% w/v, 40% w/v to 70% w/v, 50% w/v to 70% w/v. In some aspects, the first feed may comprise a calcium phosphate:synthetic polymer ratio of 1 :20. The first feed may comprise one or more biocompatible materials such as, but not limited to, calcium phosphate, synthetic polymer, chitosan, collagen, or any combination thereof.

[078] In aspects disclosed herein, the method may comprise providing a second feed to a second nozzle, wherein the nozzle extrudes a slurry comprising the second component comprising the pectin-containing sacrificial template onto the first component. In aspects disclosed herein, the second nozzle can have a nozzle size ranging from greater than 0 mm to 5 mm, such as from 0.030 mm to 4 mm, 0.030 mm to 3 mm, 0.030 mm to 2 mm, 0.030 mm to 1.8 mm, 0.030 mm to 1.6 mm, 0.030 mm to 1.4 mm, 0.030 mm to 1.2 mm, 0.030 mm to 1 mm. In certain aspects, the first nozzle can have a speed ranging from greater than 0 mm/s to 200 mm/s, such as from 5 mm/s to 200 mm/s, 5 mm/s to 175 mm/s, 5 mm/s to 150 mm/s, 5 mm/s to 125 mm/s, 5 mm/s to 100 mm/s, 5 mm/s to 75 mm/s, 5 mm/s to 50 mm/s, 5 mm/s to 25 mm/s. In particular aspects disclosed herein, the first nozzle can have a pressure of 1 bar, 2 bar, 3 bar, 4 bar, or 5 bar. In some aspects, the temperature of the second feed can have a temperature ranging from -20 °C to 100°C, such as from -10 °C to 90°C, -10 °C to 85 °C, -10 °C to 80 °C, -10 °C to 75 °C, -10 °C to 70 °C, -10 °C to 60 °C, -10 °C to 50 °C, -10 °C to 40 °C, -10 °C to 35 °C, -10 °C to 30 °C, -10 °C to 25 °C, -10 °C to 20 °C, or -10 °C to 15°C.

[079] Typically, the second component comprises pectin-containing powders, pectin-containing particulates, pectin-containing polymeric structures, or any combination thereof, which together form the pectin-containing sacrificial template that is intermittently associated with the first component. In aspects disclosed herein, the pectin concentration in the second component ranges from 5% w/v to 50% w/v, such as from 5% w/v to 45% w/v, 5% w/v to 40% w/v, 5% w/v to 35% w/v, 10% w/v to 30% w/v, 10% w/v to 25% w/v.

[080] In some aspects, a disassociating agent can be introduced to disassociate the pectin- containing sacrificial template, wherein the first component is insoluble in the disassociating agent. In certain aspects, a liquid solvent or gaseous solvent can be introduced to dissolve the pectin- containing sacrificial template. In particular aspects disclosed herein, a temperature differential can be introduced to disassociate the pectin-containing template. In a preferred aspect, the disassociating agent is an aqueous solvent.

[081] In certain aspects, the method may further comprise introducing one or more environmental modifications to provide for structural stability. In some aspects, an environment temperature can be provided such that the components of the method may comprise the environment temperature. In particular aspects disclosed herein, the method may comprise an environment temperature ranging from below 20 °C to -10 °C. In one aspect, the environment temperature of the first feed, second feed, and composition comprise an environment temperature ranging from below 20 °C to -10 °C.

[082] FIG. 1 illustrates a method 100 that includes the sequential printing of a first component 110, a second component 120 comprising a pectin-containing sacrificial template, a third component 130, and providing a disassociating agent to disassociate and remove the sacrificial template to form a three-dimensional structure 140. Moreover, FIG. 1 illustrates a three- dimensional structure 140 comprising one or more alternating layers comprising a first component 110 and a second component 120, wherein the first component comprises 110 one or more biocompatible materials and the second component 120 comprises a pectin-containing sacrificial template comprising solid particulates, polymeric structures, powders, or any combination thereof, and wherein the second component 120 is removed by disassociating from the first component via a disassociating agent (not shown) to form a three-dimensional structure 140 comprising interconnected channels and pores.

V. Overview of Several Aspects

[083] Disclosed herein is a scaffold for tissue regeneration, the scaffold comprising: a first component comprising one or more biocompatible materials; and a second component comprising a pectin-containing sacrificial template, wherein the second component is intermittently associated with the first component.

[084] In any or all aspects, the scaffold may further comprise a disassociating agent for dissolving the second component, wherein the first component is insoluble in the disassociating agent.

[085] In any or all aspects, the disassociating agent can be an aqueous solvent. [086] In any or all aspects, the one or more biocompatible materials can comprise one or more synthetic polymers, one or more calcium phosphate-containing compounds, one or more cellulose- based polymers, or any combination thereof.

[087] In any or all aspects, the one or more calcium phosphate-containing bioceramic compounds can be hydroxyapatite, oxyapatite, tetracalcium phosphate (TTCP), dicalcium phosphate anhydrous (DCPA), P-tricalcium phosphate (0-TCP), a-tricalcium phosphate (a-TCP), or calcium phosphate cement (CPC).

[088] In any or all aspects, the one or more synthetic biodegradable polymers can be polyvinyl butyral (PVB), polycaprolactone (PCL), polyvinyl alcohol (PVA), polylactic-co-glycolic acid (PLGA), or any combination thereof.

[089] In any or all aspects, the pectin-containing sacrificial template may comprise pectin methacrylate, low methoxyl pectin (LMP), high methoxyl pectin, (HMP), pectin maleate, pectin tartrate, pectin phthalate, fruit pectin, or any combination thereof.

[090] In any or all aspects, the method may comprise introducing a disassociating agent to disassociate and remove a first amount of the pectin-containing sacrificial template from the first component.

[091] In any or all aspects, the first amount of the pectin-containing sacrificial template may comprise at least 75% of a total amount of the pectin-containing sacrificial template, at least 85% of a total amount of the pectin-containing sacrificial template, or at least 90% of a total amount of the pectin-containing sacrificial template, or at least 95% of a total amount of the pectin-containing sacrificial template.

[092] In any or all aspects, the one or more biocompatible materials comprise one or more synthetic biodegradable polymers, one or more calcium phosphate-containing compounds, one or more cellulose-based polymers, or any combination thereof; and wherein the second feed comprises pectin methacrylate, low methoxyl pectin (LMP), high methoxyl pectin, (HMP), pectin maleate, pectin tartrate, pectin phthalate, fruit pectin, or any combination thereof.

[093] In any or all aspects, the first feed, second feed, and composition comprise an environment temperature ranging from below 20 °C to -10 °C. [094] Also disclosed herein is a method for making a scaffold for tissue regeneration, the method comprising: providing a first feed to a first nozzle and a second feed to a second nozzle, the first feed comprising one or more biocompatible materials, the second feed comprising pectin, pectin derivatives, or a combination thereof; and obtaining a composition comprising a first component and a second component, the first component comprising the one or more biocompatible materials, and the second component comprising a pectin-containing sacrificial template that is intermittently associated with the first component.

[095] In any or all aspects, the method may further comprise introducing a disassociating agent to disassociate the pectin-containing sacrificial template from the first component.

[096] In any or all aspects, the one or more biocompatible materials may comprise one or more synthetic polymers, one or more calcium phosphate-containing compounds, one or more cellulose- based polymers, or any combination thereof; and wherein the second feed comprises pectin methacrylate, low methoxyl pectin (LMP), high methoxyl pectin, (HMP), pectin maleate, pectin tartrate, pectin phthalate, fruit pectin, or any combination thereof.

[097] Also disclosed herein is a scaffold for tissue regeneration, the scaffold comprising a three- dimensional structure comprising one or more alternating layers comprising: a first component and a second component; wherein the first component comprises one or more biocompatible materials and the second component comprises a pectin-containing sacrificial template comprising solid particulates, polymeric structures, powders, or any combination thereof; and wherein the second component is removed by disassociating from the first component via a disassociating agent.

[098] In any or all aspects, the disassociating agent can be a liquid solvent, gaseous solvent, or an environment comprising one or more temperature differentials.

[099] In any or all aspects, the three-dimensional structure comprises adjustable dimensions and a tunable porosity with interconnected channels and pores.

[0100] In any or all aspects, the second component further comprises a water insoluble polymer and one or more active agents encapsulated by the pectin-containing sacrificial template.

[0101] In any or all aspects, the one or more active agents comprise a growth factor, a protein, an antibody, antibody fragments, peptides, oligonucleotides, plasmid DNA, plasmid RNA, or any combination thereof. [0102] In any or all aspects, one or more active agents can be controllably released by the disassociation of the pectin-containing sacrificial template.

[0103] In any or all aspects, the water insoluble polymer comprise chitosan, gelatin, alginate, or any combination thereof.

VI. Examples

[0104] Aspects of the present teachings can be further understood in light of the following examples.

Example 1

[0105] In this example, a rheological analysis of pectin ink dissolved in water at three different concentrations (10%, 17.5%, 25%) was conducted to investigate sacrificial material comprising pectin. The results of the rheological analysis are shown in FIGS. 2A-2D.

[0106] FIG. 2A is a modulus (Pa) versus shear strain (%) graph obtained from the rheological analysis of pectin slurry (pectin ink) having concentrations dissolved in water (10%, 17.5%, 25%); demonstrating that the storage modulus and loss modulus remained constant. FIG. 2B is a modulus (Pa) versus frequency (Hz) graph obtained from the rheological analysis of pectin slurry having pectin ink concentrations dissolved in water (10%, 17.5%, 25%). As shown in FIGS.2A-2B, the amplitude and frequency sweep curves, the storage modulus (G), and loss modulus (G’), remained constant for a given frequency and amplitude at the different concentrations.

[0107] Moreover, FIG. 2C is a viscosity (mPa.s) versus shear rate (1/s) graph obtained from the rheological analysis of pectin slurry (pectin ink) with concentrations dissolved in water (10%, 17.5%, 25%). In view of FIG. 2C, the shear-thinning behavior indicated that as the shear rate increased, the viscosity decreased, and therefore suitable for use in the 3D-printing process.

[0108] FIG. 2D is a modulus (Pa) versus temperature (°C) gradient graph obtained from the rheological analysis of pectin slurry (pectin ink) with concentrations dissolved in water (10%, 17.5%, 25%). Accordingly, as shown by the temperature sweep tests from 10 °C to 100 °C at 1 Hz demonstrated that G’ values were greater than G for pectin at lower temperatures; indicating a fluid-like viscoelastic behavior until reaching the crossover point at around 70 °C. Without being bound by one theory of operation, after 70 °C, G was greater than G’; thus, the hydrogels exhibited an elastic response due to water evaporation from the pectin structures similar to a solid-like structure.

[0109] In view of the results obtained from the rheological analysis, the ink temperature was maintained at 50 °C to 3D-print scaffolds comprising a pectin-containing sacrificial template in the following examples.

Example 2

[0110] In this example, scaffolds were 3D-printed using different pectin concentrations (10%, 15%, 20%, 25%) and cured with different ethanol concentrations (0%, 25%, 50%, 100%) were investigated.

[0111] FIGS. 3A-3D are images of the 3D-printed scaffolds with a 10% pectin concentration (FIG. 2A); 15% pectin concentration (FIG. 2B); 20% pectin concentration (FIG. 2C); and a 25% pectin concentration (FIG. 2D) denoted with the different ethanol concentrations (0%, 25%, 50%, 100%).

[0112] As can be seen in FIGS. 3A-3D, as the pectin concentrations and ethanol curing concentrations increased, the consistency and stiffness of the 3D structure increased and thus produced thinner filaments and stronger pectin structures. The scaffold cured in 100% deionized water (0% ethanol), however, underwent rapid degradation and dissolution. Moreover, the scaffold produced with a 25% pectin concentration and cured with 100% ethanol became stiff relative to other scaffolds.

[0113] Accordingly, this example demonstrates that by adjusting pectin and ethanol curing concentrations, scaffolds with diverse complex architectures can be 3D-printed alongside other components such as, but not limited to, hydroxyapatite (HA)/polyvinyl butyral (PVB) components; and upon dissolving in water, these structures create porous channels that can foster an environment for cell adhesion, migration, and differentiation.

Example 3

[0114] In this example, the degradation of 3D-printed scaffolds was investigated by incubating the generated scaffolds in phosphate saline buffer having a pH of 7.4 at 37 °C. The degradation kinetics are shown in FIG. 4, which show the dissociation of the pectin-containing sacrificial template (%) versus time (minutes). Table 1 shows the printing parameters for the 3D-printed scaffolds.

[0115] FIGS. 5A-5I are images of a well plate comprising a 3D-printed scaffold comprising a pectin-containing sacrificial template showing the progressive disassociation of the pectin- containing sacrificial template at 0 minutes (FIG. 5 A), 5 minutes (FIG. 5B), 10 minutes (FIG. 5C), 15 minutes (FIG. 5D), 20 minutes (FIG. 5E), 25 minutes (FIG. 5F), 30 minutes (FIG. 5G), 35 minutes (FIG. 5H), and 40 minutes (FIG. 51); demonstrating the formation of porous channels in the HA/PVB scaffold as pectin disassociates and forms the degraded scaffold.

[0116] The results obtained from the degradation kinetics (see FIG. 4) demonstrated a timedependent decrease in percentage with increased disassociation between 15 minutes and 25 minutes, which indicates the progressive disassociation of the pectin-containing sacrificial template. Moreover, complete pectin disassociation to 100% occurred at 40 minutes. Therefore, the results obtained indicate the formation of porous channels in the degraded HA/PVB scaffold as the pectin-containing sacrificial template disassociates, which can be used in applications such as, but not limited to, bone regeneration.

Example 4

[0117] In this example, the integration of the pectin-containing sacrificial template into scaffolds comprising HA and PVB having triangular, squared, circular, cylindrical, spherical, conical, and MRI/CT scanned shapes were investigated.

[0118] FIG. 6A is an image of a 3D-printed scaffold comprising a pectin-containing sacrificial template demonstrating the integration of the pectin-containing sacrificial template. FIG. 6B is an image of two 3D-printed triangular shaped scaffolds and two 3D-printed circular shaped scaffolds comprising a pectin-containing sacrificial template demonstrating the integration of the pectin- containing sacrificial template can be printed with different geometric shapes. FIG. 6C is a microscopy image of a 3D-printed scaffold comprising a pectin-containing sacrificial template showing the scaffold structure and demonstrating the integration of the pectin-containing sacrificial template. FIGS. 6A-6C demonstrate desirable precision and accuracy achieved by using a pectin- containing sacrificial template with scaffolds comprising HA and PVB.

[0119] FIGS. 6D-6E are microscopy images of a 3D-printed scaffold comprising a pectin- containing sacrificial template after the immersion in water (FIG. 6D) and after 6 hours after the immersion (FIG. 6E); demonstrating a desirable network of porous channels inside the HA/PVB scaffold generated by the dissolved pectin-containing sacrificial template.

[0120] Furthermore, FIG. 6F is a microscopy image showing a cross-sectional view of the 3D- printed scaffold comprising a pectin-containing sacrificial template post-disintegration in water and demonstrating desirable integration and subsequent pectin-containing sacrificial template of the pectin-containing sacrificial template.

[0121] This example demonstrates desirable integration and subsequent dissociation of the pectin- containing sacrificial template forms a degraded scaffold comprising a desirable architecture that supports a porous environment and facilitates cellular processes such as, but not limited to, adhesion, migration, and differentiation.

Example 5

[0122] In this example, scaffolds incorporating a pectin-containing sacrificial template arranged in three patterns were evaluated to observe structural changes before and after the dissociation of the pectin-containing sacrificial template.

[0123] FIG. 7A shows a microscopy image of a 3D-printed scaffold comprising a pectin- containing sacrificial template arranged in a linear pattern, and FIG. 7B shows the degraded scaffold after the pectin-containing sacrificial template has disassociated. These images demonstrate the formation of interconnected linear channels that facilitate nutrient and waste exchange.

[0124] FIG. 7C depicts a 3D-printed scaffold comprising a pectin-containing sacrificial template arranged in a sinusoidal pattern, while FIG. 7D shows the degraded scaffold after the after the pectin-containing sacrificial template has disassociated. The results illustrate the formation of interconnected sinusoidal channels suitable for nutrient and waste exchange.

[0125] FIG. 7E presents a microscopy image of a 3D-printed scaffold comprising a pectin- containing sacrificial template arranged in a chevron pattern, and FIG. 7F shows the scaffold after the pectin-containing sacrificial template has disassociated. These figures demonstrate the formation of interconnected chevron-shaped channels that are conducive to nutrient and waste exchange.

[0126] Accordingly, this example demonstrates that the disassociation of a pectin-containing sacrificial template arranged in linear, sinusoidal, and chevron patterns results in the formation of interconnected channels that are suitable for facilitating cellular processes essential for cell survival and tissue growth.

Example 6

[0127] In this example, the morphological characteristics of a scaffold comprising only an HA component were compared to a scaffold comprising a first component comprising HA and a second component comprising a pectin-containing sacrificial template.

[0128] FIG. 8A is a scanning electron microscope image (SEM) image showing a top view of a scaffold comprising HA constructed without a pectin-containing sacrificial template. FIG. 8B is a SEM image showing a cross view of a scaffold comprising HA constructed without a pectin- containing sacrificial template. FIGS. 8A-8B show scaffold comprising HA constructed without a pectin-containing sacrificial template exhibiting a uniform and less porous structure relative to the degraded scaffold comprising HA constructed with a pectin-containing sacrificial template and the cross-sectional view demonstrates that the first layer adhered to subsequent layers, indicating desirable interlayer adhesion.

[0129] FIG. 8C is a SEM image showing a top view of a scaffold comprising HA and a pectin- containing sacrificial template (pre-degradation). FIG. 8D is a SEM image showing a cross view of a scaffold comprising HA constructed with a pectin-containing sacrificial template (predegradation). FIGS. 8C-8D demonstrate that the incorporation of pectin into the HA scaffold resulted in a more complex surface topology, with visible pectin fibers integrated into the HA component and the cross-sectional view showed that pectin had suitable adhesion to the HA component, demonstrating desirable integration of the pectin-containing sacrificial template with the HA component.

[0130] FIG. 8E is a SEM image showing a top view of a degraded scaffold comprising HA constructed with a pectin-containing sacrificial template. FIG. 8F is a SEM image showing a bottom view of a degraded scaffold comprising HA constructed with a pectin-containing sacrificial template. FIGS. 8E-8F show that after pectin degradation, the scaffold displayed desirable changes in its architecture. The HA surface became more porous, and the cross-sectional view revealed a desirable network of interconnected channels. This transformation demonstrates that pectin degradation can facilitate mass transport and cell migration for tissue regeneration. The increased porosity and interconnected channels result in suitable nutrient and waste exchange, which support the growth of tissue.

[0131] This example demonstrates that scaffolds constructed without the pectin-containing sacrificial template (FIGS. 8A-8B) exhibit a uniform, less porous structure with interlayer adhesion. In contrast, HA scaffolds constructed with a pectin-containing sacrificial template (predegradation, FIGS. 8C-8D) display a more desirable complex surface with visible pectin fibers that are suitably integrated with the HA component. After the dissociation of the pectin-containing sacrificial template (FIGS. 8E-8F), the scaffold becomes more porous with a desirable network of interconnected channels, which facilitates mass transport and cell migration, thereby supporting tissue regeneration.

Example 7

[0132] In this example, the mechanical properties of a scaffold comprising a first component comprising HA and a second component comprising a pectin-containing sacrificial template, a degraded scaffold comprising an HA component and constructed with a pectin-containing sacrificial template, a scaffold comprising native HA, and a scaffold comprising a native pectin- containing sacrificial template were evaluated through tensile testing, measuring both strength (MPa) and Young’s modulus (5.5+1.34).

[0133] FIG. 9 is a graph showing the Young's modulus of a scaffold comprising a first component comprising HA and a second component comprising a pectin-containing sacrificial template (HA- Pectin), a degraded scaffold comprising an HA component constructed with a pectin-containing sacrificial template (HA-Degraded Pectin), a scaffold comprising native HA (HA), and a scaffold comprising a native pectin-containing sacrificial template (Pectin). FIG. 9 demonstrates a reduction in Young's modulus compared to the non-degraded scaffold, suggesting a low impact from pectin dissolution.

[0134] Table 2 shows the mechanical properties including tensile strength and Young modulus derived from the graph of FIG. 10. Table 2

[0135] The scaffold comprising an HA component and a pectin-containing sacrificial template (HA-Pectin) exhibited the highest strength (13.87+2.4 MPa) and Young’s modulus (5.5+1.34), demonstrating the synergistic interaction between HA and pectin that results in desirable mechanical stability and provides a balance between rigidity and flexibility. Following pectin dissociation, the strength decreased to 8.84+1.08 MPa, and the modulus decreased to 3.14+0.54, maintaining suitable support from the HA matrix. The native HA scaffold showed moderate values (strength: 10.84+1.42 MPa, modulus: 3.8+0.48), demonstrating HA’s inherent rigidity. In contrast, the native pectin scaffold exhibited the lowest strength (3.82+0.75 MPa) and Young’s modulus (1.75+0.134), exhibiting less rigidity.

[0136] Accordingly, this example demonstrates that incorporating a pectin-containing sacrificial template increases both the strength and stiffness of the scaffold, and even after pectin degradation, the HA component retains suitable structural integrity for tissue engineering applications.

Example 8

[0137] In this example, cell attachment of endothelial cells and osteoblasts to a scaffold comprising an HA component, a scaffold comprising an -TCP component, a degraded scaffold comprising an HA component constructed with a pectin-containing sacrificial template, and a degraded scaffold comprising a 0-TCP component constructed with a pectin-containing sacrificial template.

[0138] FIG. 10A is a bar graph showing the percentage of endothelial cells attachment to the scaffold comprising a HA component (HA), the scaffold comprising a 0-TCP component (|3-TCP), the degraded scaffold comprising a HA component constructed with a pectin-containing sacrificial template (HA-degraded Pectin), and a degraded scaffold comprising a 0-TCP component constructed with a pectin-containing sacrificial template (P-TCP-degraded Pectin) after four hours. Endothelial cell attachment was higher on the degraded scaffold comprising an HA component constructed with a pectin-containing sacrificial template (HA-degraded Pectin) (60%) relative to the scaffold comprising an HA component (HA) (20%) after four hours. The degraded scaffold comprising a P-TCP component constructed with a pectin-containing sacrificial template (0-TCP- degraded Pectin) also showed higher adhesion (60%) relative to the scaffold comprising a P-TCP component (P-TCP) alone (20%).

[0139] FIG. 1 OB is a bar graph showing the percentage of osteoblast attachment to the scaffold comprising an HA component (HA), scaffold comprising a P-TCP component (P-TCP), the degraded scaffold comprising an HA component constructed with a pectin-containing sacrificial template (HA-degraded Pectin), and the degraded scaffold comprising a P-TCP component constructed with a pectin-containing sacrificial template (P-TCP-degraded Pectin) after four hours. Osteoblast attachment was higher on the degraded scaffold comprising an HA component constructed with a pectin-containing sacrificial template (HA-degraded Pectin) (40%) compared to the scaffold comprising an HA component (HA) (20%). The degraded scaffold comprising a P- TCP component constructed with a pectin-containing sacrificial template (P-TCP-degraded Pectin) also showed higher adhesion (50%) relative to the scaffold comprising a P-TCP component (P- TCP) alone (30%).

[0140] This example demonstrates the incorporation of a pectin-containing sacrificial template into scaffolds had higher adhesion of endothelial cells and osteoblasts compared to scaffolds constructed without the sacrificial template. As such, the formation of interconnected channels through pectin degradation increased the scaffold's porosity, facilitating nutrient exchange and cell adhesion to support tissue growth and regeneration.

Example 9

[0141] In this example, the viability of endothelial cells on a scaffold comprising an HA component, a scaffold comprising an P-TCP component, a degraded scaffold comprising an HA component constructed with a pectin-containing sacrificial template, and a degraded scaffold comprising a P-TCP component constructed with a pectin-containing sacrificial template were investigating using live/dead staining.

[0142] FIG. 11A is a microscopy image showing the percentage of endothelial cell viability on a scaffold comprising an HA component using live/dead staining. The HA scaffold demonstrated endothelial cell viability of 60%, as shown by a mix of live and dead cells. The microscopy image (FIG. 11 A) shows a lower density of cells attached to the scaffold compared to the HA-degraded Pectin scaffold. Without being bound to one theory of operation, the relatively lower porosity of the HA scaffold restricted nutrient exchange and oxygen diffusion, limiting cell survival and attachment. As such, while the scaffold comprising HA alone provides structural support, its lower porosity results in less desirable conditions for endothelial cell adhesion and proliferation.

[0143] FIG. 1 IB is a microscopy image showing the percentage of endothelial cell viability on a degraded scaffold comprising an HA component constructed with a pectin-containing sacrificial template component using live/dead staining. The degraded scaffold comprising HA constructed with a pectin-containing sacrificial template exhibited higher endothelial cell viability of 80% compared to the HA scaffold alone. Without being bound by one theory of operation, the degradation of the pectin-containing sacrificial template created interconnected channels within the scaffold and therefore increasing nutrient diffusion and waste removal, which allowed for the increased cell attachment demonstrated by the higher density of live cells visible in the microscopy image. As such, the increased porosity provided a desirable environment for endothelial cell survival and therefore greater biocompatibility.

[0144] FIG. 11C is a microscopy image showing the percentage of endothelial cell viability on a scaffold comprising an 0-TCP component using live/dead staining. The scaffold comprising a 0- TCP component had an endothelial cell viability of 80% but lower than the degraded scaffold comprising a 0-TCP component constructed with a pectin-containing sacrificial template. The microscopy image shows attached cells, reflecting 0-TCP's inherent bioactivity, which promotes endothelial cell adhesion and growth. However, the relatively lower porosity of the scaffold comprising a 0-TCP component alone limits its cellular interactions compared to the degraded scaffolds constructed with a pectin-containing sacrificial template.

[0145] FIG. 1 ID is a microscopy image showing the percentage of endothelial cell viability on a degraded scaffold comprising a 0-TCP component constructed with a pectin-containing sacrificial template component using live/dead staining. The 0-TCP- degraded pectin scaffold demonstrated endothelial cell viability of 95%. The 0-TCP -degraded pectin scaffold constructed with a pectin- containing sacrificial template demonstrated higher cell attachment density and cell viability, which demonstrates more suitable biocompatibility and therefore more desirable for tissue regeneration applications. The dissociation of the pectin-containing sacrificial template increased porosity and interconnected channels within the 0-TCP matrix, facilitating nutrient exchange and oxygen diffusion. The microscopy image shows a dense population of live cells attached to the scaffold, demonstrating its ability to support endothelial cell survival and adhesion. The combination of 0- TCP's bioactivity with the pectin-containing sacrificial template makes this scaffold desirable for use in vascularization applications.

[0146] FIG. 1 IE is a bar graph showing the quantitative data of the cell viability from FIGS. 11A- 1 ID, which demonstrates that the dissociation of the pectin-containing sacrificial template in degraded HA-based scaffolds increases porosity, leading to greater cell attachment and greater viability of 80%; and the dissociation of the pectin-containing sacrificial template in degraded [3- TCP scaffolds achieved the highest endothelial cell viability of 95% due to greater nutrient and oxygen transport.

[0147] Accordingly, this example demonstrates increased scaffold porosity and cell viability on the degraded scaffolds constructed with a pectin-containing sacrificial template and are therefore more desirable in vascularization and tissue regeneration applications.

Example 10

[0148] In this example, the viability of osteoblast cells on a scaffold comprising an HA component, a scaffold comprising an [3-TCP component, a degraded scaffold comprising an HA component constructed with a pectin-containing sacrificial template, and a degraded scaffold comprising a [3-TCP component constructed with a pectin-containing sacrificial template were investigating using live/dead staining.

[0149] FIG. 12A is a microscopy image showing the percentage of osteoblast cell viability on a scaffold comprising an HA component using live/dead exhibited a lower cell density attached to the scaffold compared to the degraded scaffold comprising an HA component constructed with a pectin-containing sacrificial template. The HA scaffold exhibited osteoblast cell viability of 60%, as shown by a mix of live and dead cells. Without being bound by one theory of operation, the relatively lower porosity of the scaffold comprising HA alone lowered the nutrient exchange and oxygen diffusion, resulting in reduced cell viability relative to the degraded scaffold comprising an HA component constructed with a pectin-containing sacrificial template.

[0150] FIG. 12B is a microscopy image showing the percentage of osteoblast cells viability on a degraded scaffold comprising an HA component constructed with a pectin-containing sacrificial template component using live/dead staining. The HA-degraded Pectin scaffold demonstrated increased osteoblast viability (80%) compared to the HA scaffold. Without being bound to one theory of operation, the degradation of pectin created interconnected channels within the scaffold, for more suitable nutrient diffusion and waste removal. As such, the increased porosity facilitated greater cell attachment and survival, as demonstrated by the higher density of live cells visible in the microscopy image.

[0151] FIG. 12C is a microscopy image showing the percentage of osteoblast cell viability on a scaffold comprising an P-TCP component using live/dead staining and demonstrates that the P-TCP scaffold supported lower osteoblast viability than the degraded scaffold comprising a P-TCP component constructed with a pectin-containing sacrificial template.

[0152] FIG. 12D is a microscopy image showing the percentage of osteoblast cell viability on a degraded scaffold comprising a p-TCP component constructed with a pectin-containing sacrificial template using live/dead staining. The P-TCP-degraded pectin scaffold constructed with a pectin- containing sacrificial template exhibited the highest osteoblast viability (95%). Dissociation of the pectin-containing sacrificial template increased the porosity and interconnected channels within the P-TCP matrix, facilitating greater nutrient exchange and oxygen diffusion. Without being bound to one theory of operation, this environment supported greater osteoblast survival and attachment, as demonstrated by the dense population of live cells visible in the microscopy image.

[0153] FIG. 12E is a bar graph showing the quantitative data of the cell viability from FIGS. 12A- 12D and demonstrates that the degraded scaffold comprising an HA component constructed with a pectin-containing sacrificial template and the degraded scaffold comprising a P-TCP-degraded component constructed with a pectin-containing sacrificial template component had greater porosity, formed interconnected channels, and achieved higher cell viability.

[0154] This example illustrates that degraded scaffolds constructed with a pectin-based sacrificial template support osteoblast viability, yield higher cell density, and greater biocompatibility. In addition, their structural properties permit more desirable nutrient exchange and waste removal. Therefore, the scaffolds disclosed herein provide an environment that promotes osteoblast survival, attachment, and proliferation, making them well-suited for bone regeneration applications. Example 11

[0155] In this example, the attachment and spreading of endothelial cells and osteoblasts on the HA component were evaluated by comparing untreated HA with a scaffold comprising an HA constructed with a pectin-containing sacrificial template.

[0156] FIG. 13A is a confocal microscopy image (lOx magnification) showing endothelial cells with fluorescence staining (DAPI for nuclei and Phalloidin for actin filaments) present on a degraded scaffold comprising an HA component constructed with a pectin-containing scaffold template arranged in a line pattern. FIG. 13B is a confocal microscopy image (lOx Sora 2.8x magnification) showing endothelial cells with fluorescence staining (DAPI for nuclei and Phalloidin for actin filaments) present on a degraded scaffold comprising an HA component constructed with a pectin-containing scaffold template arranged in a linear pattern.

[0157] FIG. 13C is a confocal microscopy image (lOx magnification) showing osteoblasts with fluorescence staining (DAPI for nuclei and Phalloidin for actin filaments) present on a degraded scaffold comprising an HA component constructed with a pectin-containing scaffold template arranged in a line pattern. FIG. 13D is a confocal microscopy image (lOx magnification Sora 2.8x) showing osteoblasts with fluorescence staining (DAPI for nuclei and Phalloidin for actin filaments) present on a degraded scaffold comprising an HA component constructed with a pectin-containing scaffold template arranged in a linear pattern.

[0158] Microscopic observations in FIGS. 13A-13D demonstrate cell attachment on both surfaces; however, the HA surface of the degraded scaffold constructed with a pectin-containing scaffold template printed in a linear pattern exhibited more uniform cell adhesion and spreading.

Fluorescence staining with DAPI for nuclei and Phalloidin for actin filaments revealed a refined cytoskeletal organization and morphology on this surface. This uniformity produces a more consistent scaffold for tissue engineering. Given the roles of endothelial cells in vascularization and osteoblasts in bone formation, their integration onto the scaffolds disclosed herein is suitable for developing tissue substitutes.

[0159] While preferred aspects of the present disclosure have been shown and described herein, it will be apparent to a person of ordinary skill in the art that such aspects are provided by way of example only. Variations, changes, and substitutions to these disclosed aspects will be apparent to a person of ordinary skill in the art without departing from the present disclosure. It should be understood that all such various alternatives to the aspects described herein may be employed in practicing the present disclosure. The following claims define the scope of the disclosure.

Claims

We claim:

1. A scaffold for tissue regeneration, the scaffold comprising: a first component comprising one or more biocompatible materials; and a second component comprising a pectin-containing sacrificial template, wherein the second component is intermittently associated with the first component.

2. The scaffold of claim 1, wherein the second component is selected to be dissolved via a disassociating agent, and wherein the first component is insoluble in the disassociating agent.

3. The scaffold of claim 2, wherein the disassociating agent is an aqueous solvent.

4. The scaffold of claim 1, wherein the one or more biocompatible materials comprise one or more synthetic biodegradable polymers, one or more calcium phosphate-containing bioceramic compounds, one or more cellulose-based polymers, or any combination thereof.

5. The scaffold of claim 4, wherein the one or more calcium phosphate-containing bioceramic compounds is hydroxyapatite, oxyapatite, tetracalcium phosphate (TTCP), dicalcium phosphate anhydrous (DCPA), P-tricalcium phosphate (P-TCP), ot-tricalcium phosphate (a- TCP), or calcium phosphate cement (CPC).

6. The scaffold of claim 4, wherein the one or more synthetic biodegradable polymers are polyvinyl butyral (PVB), polycaprolactone (PCL), polyvinyl alcohol (PVA), polylactic-co- glycolic acid (PLGA), or any combination thereof.

7. The scaffold of claim 1, wherein the pectin-containing sacrificial template comprises pectin methacrylate, low methoxyl pectin (LMP), high methoxyl pectin, (HMP), pectin maleate, pectin tartrate, pectin phthalate, fruit pectin, or any combination thereof.

8. A method for making a scaffold for tissue regeneration, the method comprising: providing a first feed to a first nozzle and a second feed to a second nozzle, the first feed comprising one or more biocompatible materials, the second feed comprising pectin, pectin derivatives, or a combination thereof; and obtaining a composition comprising a first component and a second component, the first component comprising the one or more biocompatible materials, and the second component comprising a pectin-containing sacrificial template that is intermittently associated with the first component.

9. The method of claim 8, further comprising: introducing a disassociating agent to disassociate and remove a first amount of the pectin- containing sacrificial template from the first component.

10. The method of claim 9, wherein the first amount of the pectin-containing sacrificial template comprises at least 75% of a total amount of the pectin-containing sacrificial template, at least 85% of a total amount of the pectin-containing sacrificial template, or at least 90% of a total amount of the pectin-containing sacrificial template, or at least 95% of a total amount of the pectin- containing sacrificial template.

11. The method of claim 8, wherein the one or more biocompatible materials comprise one or more synthetic biodegradable polymers, one or more calcium phosphate-containing compounds, one or more cellulose-based polymers, or any combination thereof; and wherein the second feed comprises pectin methacrylate, low methoxyl pectin (LMP), high methoxyl pectin, (HMP), pectin maleate, pectin tartrate, pectin phthalate, fruit pectin, or any combination thereof.

12. The method of claim 8, wherein the first feed, second feed, and composition comprise an environment temperature ranging from below 20 °C to -10 °C.

13. A scaffold for tissue regeneration, the scaffold comprising: a three-dimensional structure comprising one or more alternating layers comprising: a first component and a second component; wherein the first component comprises one or more biocompatible materials and the second component comprises a pectin-containing sacrificial template comprising solid particulates, polymeric structures, powders, or any combination thereof; and wherein the second component is removed by disassociating from the first component via a disassociating agent.

14. The scaffold of claim 13, wherein the disassociating agent is a liquid solvent, gaseous solvent, or an environment comprising one or more temperature differentials.

15. The scaffold of claim 13, wherein the three-dimensional structure comprises adjustable dimensions and a tunable porosity with interconnected channels and pores.

16. The scaffold of claim 13, wherein the second component further comprises a water insoluble polymer and one or more active agents encapsulated by the pectin-containing sacrificial template.

17. The scaffold of claim 16, wherein the one or more active agents comprise a growth factor, a protein, an antibody, antibody fragments, peptides, oligonucleotides, plasmid DNA, plasmid RNA, or any combination thereof.

18. The scaffold of claim 17, wherein the one or more active agents are controllably released by disassociation of the pectin-containing sacrificial template.

19. The scaffold of claim 16, wherein the water insoluble polymer is chitosan, gelatin, alginate, or any combination thereof.